INTRODUCTIONBecause elements at the nanoscale behave differently than they do in their bulk form, there's a concern that some nanoparticles could be toxic. Some doctors worry that the nanoparticles are so small, that they could easily cross the blood-brain barrier, a membrane that protects the brain from harmful chemicals in the bloodstream. If we plan on using nanoparticles to coat everything from our clothing to our highways, we need to be sure that they won't poison us.
Closely related to the knowledge barrier is the technical barrier. In order for the incredible predictions regarding nanotechnology to come true, we have to find ways to mass produce nano-size products like transistors and nanowires. While we can use nanoparticles to build things like tennis rackets and make wrinkle-free fabrics, we can't make really complex microprocessor chips with nanowires yet
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Methods
Design, fabrication and cell-handling of microfluidic device for single cell electroporation
The silicon-glass microfluidic device contains two channels that are connected by microholes, which act as trapping sites for living cells (Fig 1a–b). The two microfluidic channels (widths of 50 µm and 20 µm, respectively) and the nine trapping sites (width 4 µm) are etched in the top side of a silicon wafer by reactive ion etching (RIE–depth 15 µm). Fluid reservoirs and connections to the channels are powderblasted from the backside of the silicon, followed by thermal oxidation of the silicon (318 nm) to electrically isolate it from the metal electrodes. The channels are closed with a glass wafer (Pyrex), which allows the visualization of the trapping and the electroporation process. Platinum electrodes are sputtered onto the Pyrex prior to anodic bonding to the silicon wafer with the fluidic structure, after which the wafer stack is diced into individual chips.Fig. 1 Silicon-glass microfluidic device for single cell electroporation and gene transfection studies (20 × 15 × 1 mm) (a) Artistic 3D impression of trapped cells; (b) Microfluidic chip layout, zoom-in on trapped single cells; (c) Analysis of the electric field strength distribution at the trapping sites shows that a voltage of 1 V yields an electric field strength of 0.57 kV cm–1.
The electrodes are positioned such that individual traps can be electrically addressed. The electric field is focused at the trapping sites (Fig 1c), while it is negligible in the upper channel, as well as in the neighboring trapping sites (as analyzed with finite element modeling). Thus, cells which are not trapped or trapped at the neighboring trapping sites are not affected by the electroporation protocol, as was verified experimentally.
The integrated microholes structure in the flow-through chip represents a way of handling single cells. The cell sample flows along the upper channel, while the lower channel is used to create an under-pressure, which was accomplished by suction (via a pump, 1–2 psi) on the bottom access hole. Once the cells have been trapped, the pump is switched off and cells are localized at the traps.
After trapping of cells, the DNA construct to be electroporated is loaded into the cell trap device. 40 µl of the DNA construct at a concentration of 100 ng ml–1 is added in the top and bottom reservoirs and transported into the microfluidic channels by hydrodynamic flow. DNA–cell contact is established within a pre-incubation period of 10 min before applying an electric field. Subsequently, DNA transport across the cell membrane is initiated by one electric pulse of 6 ms and electric field strength of 0.67 kV cm–1. A post-incubation time up to 10 min is given for the cell–DNA mixture before cell culture medium is added to the microfluidic channels. The chip is then immersed in a small Petri dish with this cell culture medium and placed in the incubator (37 °C and 5% CO2). Optical inspection for gene expression of the cells is done 24 h after electroporation.